Transcriptional silencing is achieved by the constitutive and coordinated modification of chromatin proteins. Sumo-targeted ubiquitin ligation (STUbL) is a newly defined biochemical activity that targets proteins for degradation by the proteasome. This activity is conserved from humans to budding yeast, and it has been shown that the human STUbL, Rnf4, can fully complement defects in the yeast mutants that are mutated in STUbL activity. Preliminary results from the Pillus lab show that yeast STUbL activity regulates transcriptional silencing and growth. Insight into this activity's role in chromatin function will come from identification of its substrates, which will serve as a platform for defining the mechanism of STUbL in chromosomal and cellular functions. The following specific aims will be carried out during the training period: (1) Identify potential STUbL substrates in existing transcriptional silencing and toxicity screens;(2) Identify STUbL substrates using proteomic approaches;and (3) Validate candidate STUbL substrates with biochemical and genetic tests. Identification and validation of targets will allow test of the hypothesis that STUbL directs degradation of proteins whose unregulated levels or polysumoylation interfere with regulation of growth and transcriptional silencing. Targeted and proteomic strategies will be used to identify potential STUbL substrates. Candidates will be subject to in-depth biochemical and genetic analyses to determine if they are indeed bona fide STUbL substrates involved in chromatin function. Experimental approaches will include mass spectrometry and immunodetection to determine if each candidate exists in a form that is both sumoylated and ubiquitinated. Genetic analysis will test null mutants of non-essential candidate genes or employ repressible promoters for essential candidates, to determine if transcriptional silencing and optimal growth in various silencing mutants is restored when degradation is achieved independently of STUbL activity. Public Health Relevance: Human Rnf4 had previously been linked to breast and prostate cancers through its interactions with estrogen and androgen receptors, and most recently, a mechanism for Rnf4's role in cancer was strengthened with the discovery that its STUbL activity functions directly in turnover of the oncoprotein PML and the aberrant PML-RARa. The proposed work to identify and validate STUbL substrates will provide insight into their roles in genomic function, as well as that of STUbL activity in transcriptional silencing and proper cell growth. The future development of therapeutic interventions to control cancer will benefit from understanding STUbl action and regulation of its substrates.